Preclinical manufacture of anti-HER2 liposome-inserting, scFv-PEG-lipid conjugate. 2. Conjugate micelle identity, purity, stability, and potency analysis.

Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 degrees C.

Sheathless electrospray ionization interfaces for capillary electrophoresis-mass spectrometric detection advantages and limitations.

A review of sheathless interfaces for capillary electrophoresis (CE)-mass spectrometry (MS) is presented. The review discusses the on-line CE-MS system requirements, advantages and weaknesses of current sheathless interface designs for CE-electrospray ionization MS, and comparison between sheath flow and sheathless interfaces. The advantages and limitations of three sheathless designs are discussed and commented upon, these include single-capillary, two-capillary and three-piece designs.

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification.

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity.

Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome.

In this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid-phase isoelectric focusing (IEF) in a novel ampholyte-free format. Twenty IEF fractions were collected and analyzed by reversed-phase microcapillary liquid chromatography (microLC)-MS/MS.

On-column sample enrichment for capillary electrophoresis sheathless electrospray ionization mass spectrometry: evaluation for peptide analysis and protein identification.

Although several designs have been advanced for coupling sample enrichment devices to a sheathless electrospray ionization-mass spectrometry (MS) interface on a capillary electrophoresis (CE) column, most of these approaches suffer from difficulties in fabrication, and the CE separation efficiency is degraded as a result of the presence of coupling sleeves. We have developed a design that offers significant improvements in terms of ease of fabrication, durability, and maintenance of the integrity of the CE-separated analyte zones. Capillaries with different inside and outside diameters were evaluated to optimize the performance of the CE-MS system, resulting in a mass limit of detection of 500 amol for tandem MS analysis of a standard peptide using a 20-microm-i.

A sheathless nanoflow electrospray interface for on-line capillary electrophoresis mass spectrometry.

A novel, rugged capillary electrophoresis-electrospray ionization (CE-ESI) interface where the separation column, an electrical porous junction, and the spray tip are integrated on a single piece of a fused-silica capillary is described. ESI is accomplished by applying an electrical potential through an easily prepared porous junction across a 3-4-mm length of fused silica. A stable electrospray is produced at nanoflow rates generated in the capillary by electrophoretic and electroosmotic forces.

Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements.

We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements. A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides. The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS.

Methods for fractionation, separation and profiling of proteins and peptides.

In the last few years there has been an increased effort to develop technologies capable of identifying and quantifying large numbers of proteins expressed within a cell system (i.e., the proteome).

Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system.

This paper presents an imaging capillary isoelectric focusing (CIEF) assay for the determination of the identity, stability, and isoform distribution of a murine monoclonal antibody (MU-B3). The experiments were conducted using a Convergent Bioscience iCE280 instrument. The optimum carrier ampholyte composition that gave the best peak separation was found to be 25% Pharmalyte pH 3-10 and 75% Pharmalyte pH 5-8.

Peptide mapping by capillary zone electrophoresis: how close is theoretical simulation to experimental determination.

A multi-variable computer model is presented for the prediction of the electrophoretic mobilities of peptides at pH 2.5 from known physico-chemical constants of their amino acid residues. The model is empirical and does not claim any theoretical dependencies; however, the results suggest that, at least at this pH, peptides may be theoretically represented as classical polymers of freely joined amino acid residues of unequal sizes.

Peptide mobility and peptide mapping in capillary zone electrophoresis. Experimental determination and theoretical simulation.

The electrophoretic mobilities of 58 peptides that varied in size from 2 to 39 amino acids and varied in charge from 0.65 to 7.82 are presented.

A simple two-dimensional high performance liquid chromatography/high performance capillary electrophoresis set-up for the separation of complex mixtures.

A two-dimensional high performance liquid chromatography/capillary electrophoresis (HPLC/CE) instrumental set-up was assembled from commercially available equipment. Fractions of the effluent from the HPLC system are collected into microtiter plates with a microfraction collector. The fractions are then dried under vacuum at room temperature, reconstituted, and analyzed by capillary zone electrophoresis (CZE).

Anti-HIV agents that selectively target retroviral nucleocapsid protein zinc fingers without affecting cellular zinc finger proteins.

Agents that target the two highly conserved Zn fingers of the human immunodeficiency virus (HIV) nucleocapsid p7 (NCp7) protein are under development as antivirals. These agents covalently modify Zn-coordinating cysteine thiolates of the fingers, causing Zn ejection, loss of native protein structure and nucleic acid binding capacity, and disruption of virus replication. Concentrations of three antiviral agents that promoted in vitro Zn ejection from NCp7 and inhibited HIV replication did not impact the functions of cellular Zn finger proteins, including poly(ADP-ribose) polymerase and the Sp1 and GATA-1 transcription factors, nor did the compounds inhibit HeLa nuclear extract mediated transcription.

Electrokinetic chromatography without electroosmotic flow.

This review summarizes the various aspects of conducting electrokinetic chromatography in coated columns with suppressed electroosmotic flow. The specific features of the technique will be presented and the potential applications explored. The equations of migration, resolution and zone spreading for neutral solutes will be presented, compared, and contrasted with those of conventional electrokinetic chromatography in bare-silica columns.

Electrokinetic chromatography in suppressed electroosmotic flow environment: use of a charged cyclodextrin for the separation of enantiomers and geometric isomers.

Electrokinetic chromatography (EKC), with negatively-charged cyclodextrins (NCDs) added to the buffer, was conducted in polyacrylamide-coated columns under suppression of electroosmotic flow. The equations of migration and resolution for neutral solutes in this mode of chromatography, which for brevity we term NCD-EKC, are presented. The chiral sulfated cyclodextrin, beta-CD-SBE (IV), used in this study is anionic over the entire pH range accessible to capillary electrophoresis, and the coated columns are stable and provide reproducible performance in the pH range 2.

Micellar electrokinetic chromatography in zero-electroosmotic flow environment.

Micellar electrokinetic chromatography (MEKC) is conducted in polyacrylamide-coated capillaries under almost complete suppression of electroosmotic flow. The equations of migration and resolution for neutral solutes in this mode of MEKC operation are presented. The technique is termed reversed-flow MEKC (RF-MEKC) because, in contrast to MEKC in bare-silica capillaries (N-MEKC), solute migration order is reversed and solute migration time is inversely proportional to micelle concentration.

Analysis of nitrate and nitrite in water and urine by capillary zone electrophoresis.

A capillary zone electrophoresis method for the separation and analysis of nitrate and nitrite in water and urine was developed. No interference in the electropherogram from other anions is observed by using a polyacrylamide-coated column with a modified phosphate buffer at pH 3 for the separation, and UV absorption at 214 nm for the detection. The method does not require sample pretreatment or the use of organic solvents.

Micellar electrokinetic chromatography of hydroxyproline and other secondary amino acids in biological samples with laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) was used for the rapid and sensitive detection of hydroxyproline in serum and hydrolyzed urine that were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC). The application of the combined o-phthalaldehyde (OPA)/FMOC derivatization in MEKC for the selective detection of secondary amino acids in biological samples is investigated.

Separation of pyridinecarboxylic acid isomers and related compounds by capillary zone electrophoresis. Effect of cetyltrimethylammonium bromide on electroosmotic flow and resolution.

The effect of the addition of cetyltrimethylammonium bromide (CTAB) to the buffer system in capillary electrophoresis on electroosmotic flow (EOF) is examined. At a CTAB concentration of 2.5 x 10(-4) M, EOF is anodal (flow towards the positive detector column end).

Laser-induced fluorescence detection of 9-fluorenylmethyl chloroformate derivatized amino acids in capillary electrophoresis.

Laser-induced fluorescence (LIF) was applied to the detection of 9-fluorenylmethyl chloroformate (FMOC-Cl) derivatized amino acids separated by capillary electrophoresis. Fluorescence excitation was provided by a pulsed, KrF laser operating at 248 nm. A limit of detection of 5 x 10(-10) M was obtained for FMOC-alanine (S/N = 2).

Optimization of resolution in capillary zone electrophoresis: combined effect of applied voltage and buffer concentration.

Expressions are formulated for the prediction of solute migration time and resolution as a function applied voltage and buffer concentration in capillary zone electrophoresis. The resolution equation assumes that solute diffusion is the only operative zone-broadening mechanism. A resolution surface in applied voltage and buffer concentration space is presented featuring isochrones that are used to predict the behavior of resolution under constant analysis time.